Spectral calibration of fluorescent polynucleotide separation apparatus

ABSTRACT

The invention relates to methods, compositions, and systems for calibrating a fluorescent polynucleotide separation apparatus. One aspect of the invention is multiple color calibration standards and their use. A multiple color calibration standard is a mixture of at least two polynucleotides of different length, wherein each of the polynucleotides is labeled with a spectrally distinct fluorescent dye. Another aspect of the invention is to produce total emission temporal profiles of multiple color calibration standards for use in calibrating fluorescent polynucleotide separation apparatus. The peaks corresponding to the fluorescently labeled polynucleotides in the total emission temporal profile may be detected using a peak detector that is driven by changes in the slopes of the total emission temporal profile. Calibration of fluorescent polynucleotide separation apparatus, with various embodiments of the methods of the invention, includes the step of identification of the labeled polynucleotides of the multiple color calibration standards. The process of spectral calibration of a fluorescent polynucleotide separation apparatus using a multiple color calibration standard may include the step of the estimating (extracting) of the dyes&#39; reference spectra, using information from the peak detection process performed on the total emission temporal profile. Other aspects of the invention include systems for separating and detecting fluorescently labeled polynucleotides, wherein the system is designed for spectral calibration in accordance with the subject calibration methods employing multiple color calibration standards. Another aspect of the invention is methods and compositions for detecting the flow of electrical current through a separation channel of a fluorescent polynucleotide separation apparatus. These methods and compositions employ monitoring dyes. Monitoring dyes are fluorescent dyes that are spectrally distinct from the dye on the polynucleotide intended to convey genetic information, e.g., fluorescent polynucleotide sequencing reaction products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/927,791, filed Aug. 10, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/154,178filed Sep. 16, 1998, both of which are incorporated herein in theirentireties by reference.

FIELD OF THE INVENTION

The invention is in the field of spectral calibration of fluorescencebased automated polynucleotide length measurement instruments.

BACKGROUND

In fluorescence-based DNA analyzers, fluorescence spectra are acquiredby exciting the sample during the analysis/assay. The information ofinterest, e.g., called bases or genotypes, is generated by transformingthe fluorescence spectra acquired during analysis/assay to “dyeamounts,” i.e., how much of each dye is present or being generatedduring the analysis/assay.

Consider, for example, the simple case of determining the amounts of twodyes present in a solution using spectral sensors. The fluorescenceemission at each spectral sensor (wavelength region or CCD bin) is thesum of the contributions of each dye. This can be expressedmathematically as:Signal at sensor i=Emission of Dye 1 at sensor i+Emission of Dye 2 atsensor i  (I)

The first thing to note about equation (I) above is that it contains oneknown quantity (measured signal at sensor i), and two unknown quantities(the emission of each dye at sensor i). Since there is one equationhaving two unknowns, no unique solution can be found. It is important tonote that including more sensors (for example a second sensor j) is notnecessarily helpful because each sensor adds an equation similar toequation (I) with two unknown quantities, namely the contributions ofthe individual dyes to the signal acquired at the sensor. In order todetermine the amounts of two dyes in a solution more information isneeded.

The additional information that enables a determination of the amountsof two dyes in a solution comes from the physical laws of fluorescenceemission. FIG. 2 shows a typical emission intensity profile as afunction of dye amount at a spectral sensor. (FIG. 2 is also referred toas the dye response function.) The segment of the dye response functionthat shows a linear relationship between the emission intensity at thespectral sensor and the dye amount is also referred to as the linearresponse range (or linear range). In FIG. 2, this range is from dyeamount=1 to dye amount=5. In practice, experimental and sampleconditions are optimized such that the analysis/assay is performed inthis range. Under these conditions, the emission of any dye at anysensor is equal to the product of the amount of dye and the slope of theresponse function in the linear range. The slope of the dye's responsefunction in the linear range is determined by the physical nature of thedye and is also known as the sensitivity. For a pure dye and a specificspectral sensor, the sensitivity is a physical constant over a givenrange of dye amounts. Equation (I) can thus be expressed as:Signal at sensor i=Ki1*A1+Ki2*A2  (II)

where Ki1 is the sensitivity of dye 1 at sensor i,

A1 is the amount of dye 1,

Ki2 is the sensitivity of dye 2 at sensor i, and

A2 is the amount of dye 2.

There are now four unknown quantities (Ki1, Ki2, A1, and A2) todetermine. Two of these unknowns (A1 and A2) depend on the sample. Theother two unknowns (Ki1 and Ki2) depend on the nature of the dye and thespectral sensors and thus can be estimated independent of the sample bywhat is referred to as spectral calibration.

Spectral calibration is thus the process by which the sensitivity ofeach dye is determined at each sensor. Doing so enables us to estimatethe parameters that are needed to analyze samples independent of thesamples. Continuing with our example of estimating the amount of twodyes in a sample in a solution, equations (3) and (4) express themeasurements acquired at two sensors i and j in relation to the dyeamounts of interest A1 and A2:Signal at sensori=Ki1*A1+Ki2*A2  (III)Signal at sensorj=Kj1*A1+Kj2*A2  (IV)

where Ki1, A1, Ki2 and A2 are as defined above (Equation (II)) and Kj1and Kj2 are the sensitivity at sensor j for dyes 1 and 2 respectively.

To determine A1 and A2 using equations (III) and (IV), we first estimateKi1, Ki2, Kj1 and Kj2 using pure dyes. Then we solve equations (III) and(IV) to estimate A1 and A2. The process of estimating Ki1, Ki2, Kj1 andKj2 using pure dyes is known as spectral calibration. The process ofusing Ki1, Ki2, Kj 1, Kj2, Signal at sensor i and Signal at sensor j toestimate A1 and A2 is known as multicomponent analysis.

Equations (III) and (IV) can be expressed in linear algebraic from as:$\begin{bmatrix}{{Signal}\quad{at}\quad{sensor}\quad i} \\{{Signal}\quad{at}\quad{sensor}\quad j}\end{bmatrix} = {\begin{bmatrix}{Ki1} & {Ki2} \\{Kj1} & {Kj2}\end{bmatrix}\begin{bmatrix}{A1} \\{A2}\end{bmatrix}}$

The matrix containing Ki1, Ki2, Kj1 and Kj2 is referred to as thecalibration matrix.

To summarize, pure dyes are used to determine the calibration matrix(Ki1, Ki2, Kj 1 and Kj2 above). This is known as spectral calibration.The calibration matrix is subsequently used to analyze samples accordingto equation (V) above.

For more details on the above background materials, see for example M.A. Sharaf, D. L. Illman and B. R. Kowalski, Chemometrics, Wiley, N.Y.,1986, Chapter 4 (p 119-p 147).

Charge Coupled Devices (CCD) can be used to detect emission spectra offluorescent dyes. A CCD-based detector can be employed in a variety ofconfigurations. For example, the CCD can be set up to cover the spectralrange of interest as an array whose elements detect discrete regions ofthe spectral wavelength range of interest. FIG. 3, for example, shows anexample of an emission spectrum (top panel, blue line), and 24 discreteregions in the wavelength domain. (top panel, red lines). Each of the 24discrete regions is referred to as a spectral bin. In this example, thewavelength range from 530 nm to 650 nm is divided into 24 spectral binsof 5 nm each.

The bottom panel of FIG. 3 represents the spectral intensities asdepicted on the CCD. The term “spectral channel” is often used to referto a “spectral bin.”

As has been discussed, spectral calibration is to estimate referencespectral profiles (reference spectra) of particular fluorescent dyesusing the optical measurement system of an automated DNA sequencer orsimilar fluorescent polynucleotide separation apparatus where theparticular dyes will be utilized. The current practice of spectralcalibration relies on measuring the spectral profile of each fluorescentdye separately. This approach to spectral calibration of fluorescentpolynucleotide separation apparatus results in reduced throughputbecause it requires N lanes on gel-based instruments and requires Nseparate runs on capillary-based instrument. As more fluorescent dyesare developed and utilized routinely (N is expected to increase), thespectral calibration of fluorescent polynucleotide separation apparatusbecomes more demanding and less efficient under the current practice.Additionally, the amount of computer resources devoted to spectralcalibration also increases with the number of dyes and separationchannels analyzed.

SUMMARY

The invention relates to methods, compositions, and systems forcalibrating a fluorescent polynucleotide separation apparatus.Fluorescent polynucleotide separation apparatus, such as an automatedDNA sequencer, must be spectrally calibrated for use with the differentfluorescent dyes to be used in conjunction with the separation system.

One aspect of the invention is multiple color calibration standards andtheir use. A multiple color calibration standard is a mixture of atleast two polynucleotide of different length, wherein each of thepolynucleotide is labeled with a spectrally distinct fluorescent dye. Ina preferred embodiment of the invention, the multiple color calibrationstandard comprises at least four polynucleotides of different length,and each of the polynucleotides is labeled with a spectrally distinctdye.

The invention includes numerous methods of spectrally calibrating afluorescent polynucleotide separation apparatus with a multiple colorcalibration standard.

Another aspect of the invention is to produce total emission temporalprofiles of multiple color calibration standards for use in calibratingfluorescent polynucleotide separation apparatus. A total emissiontemporal profile is a sum of the intensities of the fluorescence signalobtained in all spectral channels as a function of time. The peakscorresponding to the fluorescently labeled polynucleotides in the totalemission temporal profile may be detected using a peak detector that isdriven by changes in the slopes of the total emission temporal profile.Calibration of fluorescent polynucleotide separation apparatus, withvarious embodiments of the methods of the invention, includes the stepof identification of the labeled polynucleotides of the multiple colorcalibration standards. The process of spectral calibration offluorescent polynucleotide separation apparatus using a multiple colorcalibration standard may include the step of the estimating (extracting)of the dyes' reference spectra, using information from the peakdetection process performed on the total emission temporal profile.

Other aspects of the invention include systems for separating anddetecting fluorescently labeled polynucleotides, wherein the system isdesigned for spectral calibration in accordance with the subjectcalibration methods employing multiple color calibration standards.

Other aspects of the invention include systems for separating anddetecting fluorescently labeled polynucleotides, wherein the system isdesigned for spectral calibration in accordance with the subjectcalibration methods employing multiple color calibration standards. Thesubject systems comprise a fluorescent polynucleotide separationapparatus and a computer in functional combination with the apparatus.

Another aspect of the invention is methods and compositions fordetecting the flow of electrical current through a separation channel ofa fluorescent polynucleotide separation apparatus. These methods andcompositions employ monitoring dyes. Monitoring dyes are fluorescentdyes that are spectrally distinct from the dye on the polynucleotideintended to convey genetic information, e.g., fluorescent polynucleotidesequencing reaction products.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is diagram of an example of a portion of a temporal profilelabeled as to show examples of some of the terms used herein.

FIG. 2 shows a typical response function of a dye at a spectral sensor.

FIG. 3 shows an example of an emission spectrum (top panel, blue line),and 24 discrete regions in the wavelength domain (top panel, red lines).Each of the 24 discrete regions is referred to as a spectral bin. Inthis example, the wavelength range from 530 nm to 650 nm is divided into24 spectral bins of 5 nm each.

FIG. 4 illustrates a data flow scheme, according to an embodiment of thepresent invention.

DEFINITIONS

The term “fluorescent polynucleotide separation apparatus” as usedherein denotes an apparatus for separating fluorescently labeledpolynucleotide mixtures (e.g., by electrophoresis) and detecting theseparated polynucleotides by the fluorescence emission produced fromexciting the fluorescent dye. Examples of fluorescent polynucleotideseparation apparatus include automated DNA sequencers such as the PEApplied Biosystems 310 and 377 (Foster City, Calif.). Examples offluorescent polynucleotide separation apparatus are also described in,among other places, U.S. Pat. Nos. 4,971,677; 5,062,942; 5,213,673;5,277,780; 5,307,148; 4,811,218; and 5,274,240. The term fluorescentpolynucleotide separation apparatus also includes similar instrumentsfor polynucleotide fragment length analysis that are not capable of thesingle base pair resolution required to obtain DNA base sequenceinformation. Fluorescent polynucleotide separation apparatus comprisesone or more separation regions or channels, typically the path ofelectric current flow in electrophoretic separation devices. Types ofseparation channels include capillaries, microchannels, tubes, slabgels, and the like. Fluorescent polynucleotide separation apparatuscollect several types of data during their operation. This data includesspectral data and temporal data relating to the fluorescent labeledpolynucleotides separated by the apparatus. Typically, such data iscollected by a detector (e.g. a CCD array, photomultiplier tubes, andthe like) designed to obtain quantitative spectral data over apredetermined region or regions of the separation channels. Spectraldata collected by the apparatus includes the intensity of fluorescenceat a plurality of wavelengths. The different wavelengths sampled arereferred to as bins or channels. The apparatus also collects temporaldata that is correlated with the spectral data. The temporal data iscollected at numerous different time points. For example, a detector ata fixed position will measure increases and decreases in fluorescenceintensity as a function of time as a labeled polynucleotide peak passesby the detector. This temporal data may be expressed as “frame” or“scan” number to indicate the different temporal sampling points.

A temporal profile is a plot of the intensity of a spectral signal as afunction of time or scan/frame number. A temporal profile consists ofsystematic and random variations. Systematic variations are caused bypeaks, spikes and background drifts. These variations cause the shape ofthe profile to undergo specific, and often predictable, changes. Bycontrast, random variations do not cause specific or predictable changesin the temporal profile. A temporal profile has segments that correspondto baseline (baseline segment) and segments that correspond to peaks(peak segments), and segments that correspond to spikes. Baselinesegments are made of random variations superimposed on offset value(s).

An emission temporal profile is a plot of the intensity of the signalsobtained in a certain spectral channel/bin as a function of time orscan/frame number.

A total emission temporal profile is a plot of the sum of theintensities of the signals obtained in all spectral channels/bin as afunction of time or scan/frame number.

The analytical background of a temporal profile is the average of thesignals obtained along a segment of the profile where the segment isvoid of peaks, spikes and systematic variations (i.e., a baselinesegment.) This is schematically shown in FIG. 1. The analytical noise ofa temporal profile is the standard deviation of the signals obtainedalong a segment of the profile where the segment is void of peaks,spikes and systematic variations. Analytical background and noise maychange as a function of time along the temporal profile. This occurswhen there are drifts in the background.

The term net analytical signal refers to the intensity at any point of aprofile after correcting for background and baseline offsets and/ordrifts. The analytical signal to noise ratio (S/N) is the ratio of thenet analytical signal to the analytical noise. Net analytical signalsmay, or may not, be significant depending on their S/N's.

A peak detector is a mathematical transformation of a profile (e.g., atemporal profile) whose purpose is to locate peaks along the profile. Apeak detector is defined by the type of the transformation, and thedetection parameters associated with its operation. A typical peakdetector distinguishes between segments of a profile that representbaseline (an offset with random noise) and other segments that representpeaks and spikes based on the slope of the temporal profile. From thepeak detector's point of view, a baseline segment is a set of datapoints along the temporal profile where the absolute value of the slopeof the profile does not exceed the peak detector's threshold. An idealpeak detector ignores baseline and spike segments, and retainsinformation relevant only to peaks (in our case the componentpolynucleotides of the multiple color calibration standard.)

Peak slope threshold is a value which if exceeded by the slope of atemporal profile, the presence of a potential peak is indicated. Thisvalue may be referred to as the “threshold” parameter of the peakdetector. If a peak is actually present, the threshold value is alsoused to indicate that the temporal profile has returned to baselinelevels and that the peak has ended.

Peak start is the first point along the peak segment of a temporalprofile. A peak start may be found at baseline levels, or in the valleybetween two peaks. Peak end is the last point along the peak segment ofa temporal profile. A peak end may be found at baseline levels, or inthe valley between two peaks. Peak maximum is a point along the peaksegment of a profile where the highest intensity is found. Peak width isthe number of data points between the start of the peak and the end ofthe peak (see FIG. 1.) The peak width attribute is helpful indiscriminating between peaks that correspond to labeled DNA fragmentsand spikes. The latter have relatively smaller peak widths.

Peak height at maximum is the intensity at peak maximum corrected forthe analytical background (see FIG. 1.) Peak S/N ratio refers to theratio of the peak height at maximum to the analytical noise of thetemporal profile. A peak's S/N attribute is an effective parameter thatis used to retain the peak information of the dye-labeled fragments ofthe multiple color calibration standard.

Migration time of a peak is the time elapsed from the start of theelectrophoresis to peak maximum. A particular peak corresponding to acertain labeled polynucleotide of the multiple color calibrationstandard may serve as a reference peak whose migration time is areference point from which the migration time of other peaks aremeasured.

Migration time offset is the difference between the migration time of aparticular peak and the migration time of the reference peak (see FIG.1.) Peaks to the left of the reference peak will have negative migrationtime offsets, while those to the right of the reference peak will havepositive migration time offsets. Reference peaks are located based onrank or migration time. Subsequently, migration time offsets are used tolocate all other dye-labeled fragments.

Input parameters are attributes that are used by a particularimplementation of the algorithm. These parameters may be specific to themultiple color calibration standard as well as to the platform beingused. The implementation attributes may include the peak width, thethreshold variable, the peak S/N ratio, the reference peak locator(migration time vs. rank), the migration time offsets, and theappropriate tolerances, if necessary, to account for instrumental andexperimental variations.

The term “polynucleotide” as used herein refers to naturally occurringpolynucleotides such as DNA and RNA and to synthetic analogs ofnaturally occurring DNA, e.g. phosphorothioates, phosphoramidates,peptide nucleic acids (PNAs), and the like. The term “polynucleotide”does not convey any length limitation and should be read to include invitro synthesized oligonucleotides.

Specific Embodiments Of The Invention

The fluorescence spectra that are acquired during a sequencing reactionor a homogenous assay are typically mixture spectra originating fromco-migration of DNA fragments with different dye labels (e.g., in thecase of sequencing) or the utilization of multiple probes with differentdye labels (e.g., in the case of homogeneous assays). In order todetermine the type and amount of each dye being detected, the acquiredmixture spectra need to be decomposed such that the contribution of eachdye is estimated. In order to do so, one needs to measure the emissionspectrum of each pure dye. The process of estimating the spectralprofile of each pure dye is often referred to as “spectral calibration”.Once the spectral profile of each of the pure dyes is estimated, one cananalyze mixture spectra and estimate the contribution of each dye beingdetected. This process (analyzing mixture spectra associated withsamples and assays) is generally known as “multicomponent analysis.”(See, e.g., J. Yin et al., “Automated Matrix Determination in Four DyeFluorescence-Based DNA Sequencing,” Electrophoresis 17:1143-1150 (1996);W. Huang et al., “A Method to Determine the Filter Matrix in Four-DyeFluorescence-Based DNA Sequencing,” Electrophoresis 18:23-25 (1997); K.M. O'Brien et al., “Improving Read Lengths by Recomputing the Matricesof Model 377 DNA Sequencers,” BioTechniques 24:1014-1016 (1998); and“User Bulletin, Making a Matrix”, PE Applied Biosystems (1996); each ofwhich is incorporated herein by reference.

The invention relates to methods, compositions, and systems forcalibrating a fluorescent polynucleotide separation apparatus.Fluorescent polynucleotide separation apparatus, such as an automatedDNA sequencer, must be spectrally calibrated for use with the differentfluorescent dyes to be used in conjunction with the separation system.Spectral calibration may also be used to account for variations betweenindividual fluorescent polynucleotide separation apparatus and accountfor changes that occur in a given instrument over time. Fluorescent dyeshave characteristic emission spectra for a given excitation wavelength.When multiple different dyes are present in a mixture for separation,the individual contributions of the different dyes to a spectraldetection reading must be separated from one another. Such separationmay be achieved through the use of a matrix containing spectral emissiondata of the various dyes used for analysis, see Yin et al.,Electrophoresis 17:1143-1150 (1996) and U.S. patent application Ser. No.08/659,115, filed Jun. 3, 1996. The generation of a spectral calibrationdata matrix for calibrating a fluorescent polynucleotide separationapparatus typically includes the steps of introducing a fluorescentpolynucleotide calibration standard into a fluorescent polynucleotideseparation apparatus, separating the labeled polynucleotides from eachother, and detecting the separated polynucleotides with a detector. Thedetector collects spectral information relating to the concentration oflabeled polynucleotides at a specific location (or locations) on theapparatus. The information collected is the fluorescent emissions at aplurality of wavelengths, (e.g., bins/channels). The informationobtained by the detector includes the recording of temporal data (e.g.scan number, for a fluorescent polynucleotide separation apparatus thatemploys a scanning detector) correlated with the spectral emission datafor the measured time points.

One aspect of the invention is to produce total emission temporalprofiles of multiple color calibration standards for use in calibratingfluorescent polynucleotide separation apparatus. A total emissiontemporal profile is a sum of the intensities of the fluorescence signalobtained in all spectral channels as a function of time. Peakscorresponding to the different oligonucleotides in the multiple colorcalibration standard may then be determined by analyzing the totalemission temporal profile with a peak detection transformation function.A reference spectrum for each of the fluorescent dyes of interest usedin the multiple color calibration standard may then be produced byselecting a reference spectrum that substantially corresponds to therelevant peak of the total emission profile.

Other aspects of the invention are multiple color calibration standardsand their use. A multiple color calibration standard is a mixture of atleast two polynucleotides of different length. (It will be understood bypersons skilled in the art that each polynucleotide is present in alarge number of essentially identical copies so as to provide usefulamounts of the subject compositions) Preferably, the length (in numberof bases) of each labeled polynucleotide is known precisely so as tomaximize the accuracy of the standard. Each of the different lengthpolynucleotides in the standard is labeled with a different fluorescentdye. The predetermined correlation between the length of the givenpolynucleotide and the particular fluorescent dye that is attached tothat polynucleotide is used to identify the polynucleotide of themultiple color calibration standard during the calibration process. Thedifferent fluorescent dyes are selected so as to have distinctivespectral profiles (for the same excitation frequency). Preferably thesizes of the polynucleotides in the multiple color calibration standardare selected so as to ensure sufficient separation between thepolynucleotides labeled with different dyes such that the spectralprofile peaks of the fluorescent dyes do not significantly overlap. Inother words, there is preferably sufficient difference between thelengths of the constituent polynucleotides so that for any givenpolynucleotide peak that is being detected, the possibility that thefluorescence intensity readings are the result of multiple differentdyes is minimal.

The sizes of the polynucleotides that are in multiple color calibrationstandards are selected so as to be within the size separation for theparticular fluorescent polynucleotide separation apparatus for whichthey are designed to be used. Exemplary of such a range is about 10-1500bases in length, preferably about 10-1000 bases in length, morepreferably about 20-500 bases in length. Preferably polynucleotides inthe standard are separated by at least 10 bases in length. Methods ofmaking the polynucleotide components of the subject standards are wellknown to persons of ordinary skill in the art. Such methods include thecomplete in vitro synthesis of the polynucleotide, e.g. through the useof phosphoramidite chemistry. Alternatively, the polynucleotides may besynthesized enzymatically. For example a PCR (polymerase chain reaction)amplification may be performed using primers separated by the desireddistance, wherein one of the amplification primers is labeled with afluorescent dye of interest.

In preferred embodiments of the invention, the multiple colorcalibration standard comprises at least four polynucleotides ofdifferent length, and each of the polynucleotides is labeled with aspectrally distinct dye. The use of four spectrally distinct dyes, eachbeing essentially the same as the dyes used for producing polynucleotidesequencing reaction products is of particular interest for use in fourcolor chain termination type sequencing (employing either fluorescentlylabeled chain terminating nucleotides or fluorescently labeled primers).The multiple color calibration standard may comprise one or morefluorescent dyes in addition to the dyes in the standard that correspondto the dyes used in sequencing reactions that are designed for use inconjunction with the particular standard. These additional dyes may be“signal dyes” as described later in this application. These additionaldyes, which are preferably attached to polynucleotides, may be used tomonitor the electrical current flow through the separation channel orchannels of a fluorescent polynucleotide separation apparatus. Whiledetection of electrical current flow through a fluorescentpolynucleotide separation apparatus without the use of additional dyesis relatively simple for apparatus employing a single separationchannel, e.g. a slab gel, the detection of current through amulti-channel system, e.g., a multiple capillary system, is difficultwithout using additional dyes. The movement of these additional dyes,which should also be added to the sample for analysis, through thefluorescent polynucleotide apparatus may be detected in order to verifythe flow of electrical current through a separation channel, e.g., anindividual capillary.

The invention also includes kits for performing the subject method. Thekits comprise the individual fluorescently labeled polynucleotidecomponents of the subject multiple color spectral calibration standards.By providing the individual components of a standard, end users mayconveniently produce their own standard for specific applications.

A wide variety of florescent dyes may be used to label thepolynucleotides in multiple color calibration standards. Fluorescentdyes are well known to those skilled in the art. Examples of fluorescentdyes include fluorescein, 6-carboxyfluorescein,2′,4′,5′,7′-tetrachloro-4,7-d-ichlorofluorescein,2′,7′-dimethoxy-4′,5′-6-carboxyrhodamine (JOE),N′,N′,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) and6-carboxy-X-rhodamine (ROX). Fluorescent dyes are described in, amongother places, U.S. Pat. No. 4,855,225; Menchen et al, U.S. Pat. No.5,188,934; Bergot et al, International Application PCT/U.S. Pat. No.90/05565; Haugland, R. P., Handbook of Fluorescent Probe and ResearchChemicals, 6th edition (1996) and like references. Methods of attachingfluorescent dyes to polynucleotides are also well known to those skilledin the art. Examples of such attachment methods can be found in, amongother places, U.S. Pat. Nos. 4,789,737; 4,876,335; 4,820,812; and4,667,025.

The multiple color calibration standards of the invention may alsocomprise various other components in addition to fluorescent labeledpolynucleotides. Such additional components may be used to improve themovement of the polynucleotide through a separation channel of afluorescent polynucleotide separation apparatus. Examples of additionalcomponents include, but are not limited to, buffers, denaturants, andthe like.

The invention includes numerous methods of spectrally calibrating afluorescent polynucleotide separation apparatus with a multiple colorcalibration standard. A multiple color calibration standard isintroduced, i.e., loaded, into a fluorescent polynucleotide separationapparatus. The introduction of a multiple color calibration standardinto a florescent polynucleotide separation apparatus and the subsequentseparation of the components of the standard along with the collectionof the spectral and temporal data obtained from detecting the separatedlabeled polynucleotides may be conveniently referred to as producing aspectral calibration run. Spectral calibration runs may be performed ona single separation channel or may be simultaneously performed onseveral separation channels.

A spectral calibration run produces data that can conveniently beanalyzed in the form of a matrix, D, with R rows and C columns, thatcontains the measured intensities in each spectral channel/bin (thecolumns of the data matrix) as a function of time or frame/scan number(the rows of the data matrix). Each of the C columns represents anemission temporal profile for the corresponding spectral channel/bin.Each of the R rows represent the spectrum acquired during thecorresponding data collection/acquisition period. The person of skill inart may devise numerous equivalent representations of the data obtainedfrom a calibration run rather the specific matrix described above, e.g.the components of the rows and columns may be transposed or the data maybe manipulated without the use of a 2-D matrix. Each temporal profilecontains peaks of different shapes that correspond to the dye-labeledpolynucleotides of the multiple color calibration standard. The shape ofeach of these peaks depends on the emission characteristics of thecorresponding dye at the specific spectral channel/bin represented bythe temporal profile. A total emission temporal profile may then beprepared by summing the intensities of the signals obtained for allspectral channels/bins as a function of the temporal parameter, e.g.scan/frame number. Ideally, the emission temporal profiles for thelabeled polynucleotides of a multiple color spectral calibrationstandard are “parallel.” In practice, however, this ideal property mayshow deviations that are caused by heterogeneous emission efficiencies,baseline drifts, minor spectral measurements anomalies and deviationsfrom the analytical linear dynamic range. Despite sharing importantgeneral properties (peaks of multiple color spectral calibrationstandard constituent labeled polynucleotide separated by baselinesegments,) the temporal profiles of the individual spectralchannels/bins may exhibit large variations in S/N ratios, noisedistribution as well as peak shapes. In order to minimize such problems,total emission temporal profiles may be used for calibration rather thanindividual emission temporal profiles. An advantage of total emissionprofiles is the inclusion of all polynucleotide components of thestandard regardless of differences in emission intensities between thespectral channels/bins. The total emission profile, thus, provides atemporal profile that contains all the peaks of the multiple colorspectral calibration standards labeled polynucleotide, and only one setof detection input parameters is necessary.

The peaks corresponding to the fluorescently labeled polynucleotide inthe total emission temporal profile may be detected using a peakdetector that is driven by changes in the slopes of the total emissiontemporal profile. When the slope of the total emission temporal profileexceeds a certain threshold, the start of a potential peak is detected.The potential peak may then be traced through its crest/maximum anduntil the potential peak ends by either having the total emissiontemporal profile returns to background levels, or detecting the start ofanother peak. The information regarding the start, maximum and end ofthe potential peak may then be evaluated to assess the significance ofthe peak. Only significant peaks (in terms of the minimum requirementsindicated by the peak width and peak S/N ratio input parameters) areused to select reference spectra. This process may be used to rejectspikes and insignificant/non-target peaks while retaining the peakscorresponding to the components of the multiple color calibrationstandard.

Peak Detection Transformation

Peak detection is performed on a total emission temporal profile. Apreferred transformation to detect peaks is the slope of the totalemission temporal profile, and is given as:S _(i)=(I _(i+1) −I _(i))+(I _(i+2) −I _(i−1))  (1)

where S_(i) is the slope (as estimated by the detection transformation)at point i, and I_(k) is the intensity of the total emission temporalprofile at point k. However, other peak detection transformations basedon changes of intensity may also be used in the subject methods.

Statistical Distribution Of Detection Transformation And FailureAnalysis

The threshold parameter used in a peak detector may be an actual valuefor the slope. However, in a preferred embodiment of the invention thethreshold is determined by the distribution of the peak detectiontransformation based on a probabilistic model. An input variable is usedto estimate the threshold. The detection transformations produce aparameter, for example S in Equation 1, that is used for peak detection.The performance of S in distinguishing baseline segments from peaksegments in a temporal profile is highly influenced by the distributionof S when I is subjected to random variations only. The variance in Scan be estimated by applying error propagation theory to Equation 1, andis given according to:σ²(S)=Σ{[∂F(S)/∂I _(k)]²σ²(I _(k))}

where F(S) is the detection transformation (Equation 1). For independentmeasurements, the above expression reduces to:σ²(S)=4σ²(I)  (2)

Thus, segments of a temporal profile that correspond to baselines withrandom variations are expected to produce amplified variations,according to Equation 2, after the detection transformation.

The start of a peak is considered the first data point along the peaksegment of the total emission profile that does not belong in thebaseline population. The baseline segment's population produces atransformation distribution with a variance of 4σ²(I) (Equation 2). TheS distribution's variance can, therefore, be used to set a detectionthreshold with a probability of failure (incorrectly classifying a datapoint from the baseline population as the start of a peak segment) thatis given asPr[|S _(i−)μ(S)|≧kσ(S)⁻ ]≦k ⁻²  (3)

where μ(S) is the mean of the S distribution, and is expected to bezero.

For example if the threshold is set at 3σ(S), the probability ofselecting a data point from the baseline segment's population as a peakstart is, according to Equation 3, 100/9 or about 11%. (Equation (3)does not assume a Gaussian, or any other, distribution of the baselinedata points population.) To decrease the probability of failure, thethreshold may be increased, or one may consider the peak start as twoconsecutive data points whose transformation exceeds the thresholdvalue. If the threshold is set, again, at 3σ(S), the probability ofS.sub.iexceeding this value at two consecutive measurements when onlyrandom variation are present is about 1%. The peaks corresponding to thelabeled polynucleotides of the multiple color calibration standards areexpected to be among the peaks with the highest peak S/N ratios. Sinceall detected peaks may be subjected to additional criteria such asminimum peak S/N ratio and minimum peak width, false peak starts(detected with a probability of 1% as outlined above) are not expectedto cause any significant problems in detecting and retaining the peakscorresponding to the labeled polynucleotides of the multiple colorcalibration standards while rejecting spikes and other non-target peaks.

The outcome of the peak detection process is a set of attributes for allpeaks that satisfy the minimum peak width and the minimum peak S/N ratiorequirements. This information includes the data point at the start ofthe peak, the data point at the end of the peak. Appropriate descriptorsindicating whether the peak start point is at baseline levels or in avalley between two peaks are also compiled during the peak detectionprocess. Similarly, peak end points are flagged as either being atbaseline levels or in a valley between two peaks. Peak information alsoincludes the data point at which the peak maximizes, and the intensityat the peaks' maxima as well as the actual peak width. Where available,the locations of baseline segments to the left of the peak start and tothe right of the peak end may also be compiled.

Identification Of The Components Of Multiple Color Calibration Standards

Calibration of fluorescent polynucleotide separation apparatus withvarious embodiments of the methods of the invention include the step ofidentification of the labeled polynucleotides of the multiple colorcalibration standards. The identification of the colored ladderfragments refers to the assignment of each labeled polynucleotide in amultiple color calibration standard to one of the peaks retained by thepeak detector. Assignment can be accomplished by a variety of methods.Since the spectral calibration of fluorescent polynucleotide separationapparatus is accomplished under controlled conditions (known andprespecified materials and experimental parameters), an efficient way toidentify the labeled polynucleotides of the multiple color calibrationstandards is to take advantage of the controlled experimental conditionsand the design of the colored ladder. For example, the multiple colorspectral calibration standard design may be such that the fragmentlabeled with the dye DR110 in a multiple color calibration standard hasthe largest migration time. Under optimized and controlled experimentalconditions, where the peak width and peak S/N ratio parameters allowmultiple color calibration standard constituent polynucleotides to bedetected and retained, the last peak would be the DR110-labeledfragment. A peak with such a high probability of being detected mayserve as a reference peak to locate peaks corresponding to the otherlabeled polynucleotides of the multiple color calibration standard.Since the migration of a labeled DNA fragment is influenced primarily bythe size of the DNA fragment, the labeling dye and the separationmatrix, migration time offsets over a short migration interval areeffective parameters to use in locating the peaks corresponding to thelabeled polynucleotides of the multiple color calibration standardsgiven the location of a reference peak such as the DR110-labeled peak.

If the mobilities of the labeled polynucleotides of the standard exhibitsignificant nonlinearities, and the migration of the colored ladderfragments is not easily (and reliably) predictable over a large range ofmigration times using offsets from one reference peak, the predictionrange may be reduced by relying on offsets from neighboring peaks. Forexample, a polynucleotide labeled with DR110 may be used as a referencepeak to locate the polynucleotide (in the same multiple colorcalibration standard mixture) labeled with DR6G. Subsequently, thepolynucleotide labeled with DR6G (in the same standard) may serve as areference peak to locate the polynucleotide labeled with DTAM. Thepolynucleotide labeled with DTAM (in the same standard) may then used tolocate the polynucleotide labeled with DROX. Finally, the polynucleotidelabeled with DROX (in the same standard) may serve as a reference peakto locate the polynucleotide labeled with JAZ.

Peak Detection Parameters

The input parameters of labeled polynucleotides of the multiple colorcalibration standards for peak detectors may include, but are notlimited to:

(a) The starting point and the sample size to be used in estimating theanalytical background and the analytical noise in the total emissiontemporal profile (σ(I) in Equation 2.) The analytical background andnoise are used to assess the peak S/N ratio.

(b) The threshold variable corresponding to k in Equation 3. Thisdetermines the sensitivity of the peak detector to baseline variations.

(c) The threshold variable to be used in detecting baseline segments tothe left of peak starting points and to the right of peak ending points,where available. Typically, this is a value less than that used fordetecting peak starting points

(d) Minimum peak width and peak S/N ratio requirements. These twoparameters are selected such that spikes and non-target peaks areignored. Ideally, only the peaks corresponding to the fragments of thecolored ladder are retained by the peak detector.

(e) Reference peak migration time and its tolerance. If this parameteris zero, the last peak found is by default the reference peak.

(f) Migration time offsets of the colored ladder fragment peaks andtheir tolerances.

(g) The appropriate search windows for maxima and baseline values forthe emission temporal profiles.

(h) Number of the colored ladder fragment peaks and the maximum numberof peaks expected to be found in the total emission temporal profile.These parameters are used for memory management.

Estimation Of Dyes' Reference Spectra

The process of spectral calibration of fluorescent polynucleotideseparation apparatus using multiple color calibration standard mayinclude the step of the estimating (extracting) of the dyes' referencespectra from the acquired data matrix, D, using information from thepeak detection process. As stated earlier, the rows of the data matrix,D, contain the spectral information. Any spectrum acquired during anydata collection/acquisition period can be estimated from the netanalytical signals obtained in the spectral channels/bins. A spectrumis, thus, a background/baseline corrected row of D.

The dyes' reference spectra are, therefore, estimated from the correctedrows of D that correspond to data points along the peak segments of thetotal emission temporal profile. The peak maximum is the data point (rowof D) recommended for estimating the dyes' reference spectra. Since theemission temporal profiles of the individual spectral channels/bins arenot expected to be perfectly parallel, a row of D is corrected byestimating the net analytical signal in each spectral channel/bin usingthe peak detection information from the total emission temporal profileand appropriate search windows. Spectral calibration reference spectraare, also, normalized such that the maximum spectral intensity in eachspectrum is set to equal 1. This is accomplished by dividing allcorrected spectral intensities in each spectrum by the maximum correctedspectral intensity found in the spectrum.

-   -   Uncertainties In Dyes' Reference Spectra The spectral intensity        in a particular channel/bin of a normalized dye's reference        spectrum can be expressed as:        R _(i) =I _(i) /I _(m)  (4)

where R_(i) is the normalized spectral intensity in the referencespectrum at the ith spectral channel/bin,

I_(i) is the net analytical signal in the ith spectral channel/bin, andI_(m) is the highest net analytical signal in the spectrum.

The uncertainty in R_(i), is given according to:σ²(R _(i))/R _(i) ²=(σ² /I _(i) ²)[1+m ²]  (5)

where m is given as I_(i)/I_(m′)and σ² the variance in the spectralintensities and is assumed to be equivalent in both spectralchannels/bins.

The relative error in R_(i) may be expressed according to:σ(R _(i))/R _(i)=[1/SNR _(i)][1+m ²]^(1/2)  (6)

where SNR_(i) is the signal-to-noise ratio of the net analytical signalin the ith spectral channel/bin.

The term [1+m²] in Equations 5 and 6 never exceeds the value of 2according to the normalization defined by Equation 4. The relative errorin R_(i) can, therefore, be expressed as:σ(R _(i))/R _(i)≦[1/SNR _(i)]^(✓)2  (7)

where SNR_(i) is the signal-to-noise ratio of the net analytical signalin the ith spectral channel/bin.

The analytical implication of Equation 6 (and Equation 7) is that thequality of the dyes' reference spectra increases (i.e., the relativeerrors in the spectral bins decreases) as the signal-to-noise ratio ofthe net analytical signal increases. The reliability of spectralestimation is determined primarily by the signal-to-noise ratio, not bythe number of spectra being used to obtain an average estimate. Sincethe spectra acquired at peaks' maxima have the highest S/N ratio, thesespectra are the preferred spectra to be selected as reference spectra asthey are expected to have the lowest relative errors. However, otherspectra that substantially correspond to the peak maxima may also beused as reference spectra.

Other embodiments of the invention include systems for separating anddetecting fluorescently labeled polynucleotides, wherein the system isdesigned for spectral calibration in accordance with the subjectcalibration methods employing multiple color calibration standards. Thesubject systems comprise a fluorescent polynucleotide separationapparatus and a computer in functional combination with the apparatus.The term “in functional combination” is used to indicate that data fromthe fluorescent polynucleotide separation apparatus, such data includingfluorescence intensity data over a range of detection wavelength and theassociated temporal data, is transferred to the computer in such a formthat the computer may use the data for calculation purposes. Thecomputer in the system of the invention is programmed to perform thespectral calibration method of the invention using the data producedfrom running a multiple color spectral calibration standard. Thus thecomputer is programmed to produce a total emission temporal profile fromthe spectral and temporal data obtained from the calibration run. Thecomputer may also be programmed to detect peaks in the total emissiontemporal profile, and determine reference spectral profiles of the dyesattached to the labeled polynucleotide represented by the peaks. A widevariety of computers may be used in the subject system. Typically, thecomputer is a microprocessor and the attendant input, output, memory,and other components required to perform the necessary calculations. Thecomputers may be generally programmable so as to facilitatemodifications or the apparatus of the computer program may be in theform of “firmware” that is not readily subjected to modification.

Other embodiments of the invention include systems for calibrating afluorescent polynucleotide separation apparatus. The calibration systemsinclude computer code that receives a plurality of spectral and temporaldata from a fluorescent polynucleotide separation apparatus. The systemalso comprises computer code that calculates a total emission temporalprofile from the spectral and temporal data. The system may furthercomprise additional computer code for performing the subject methods ofspectral calibration. Such additional code includes code for detectingpeaks, and code for preparing a spectral profile for each of the dyesincluded in a calibration standard. As the computer code of the subjectsystem requires a physical embodiment to function, the system alsocomprises a processor and computer readable medium (e.g. optical ormagnetic storage medium) for storing the computer program code. Thecomputer readable medium is functionally coupled to the processor.

Another aspect of the invention is methods and compositions fordetecting the flow of electrical current through a separation channel ofa fluorescent polynucleotide separation apparatus. Such methods andcompositions are particularly useful with fluorescent polynucleotideseparation apparatus that employ multiple separation channels, e.g. amulti capillary or multiple microchannel system, because ofinterruptions in current flow in individual separation channels may bedifficult to detect if a substantial percentage of the channels haveproper current flow. The subject electrical flow monitoring methodsinvolve the use of fluorescent dyes that are spectrally distinct fromfluorescently labeled polynucleotides of primary interest. Thesespectrally distinct fluorescent dyes are referred to herein asmonitoring dyes. In a preferred embodiment of the invention, themonitoring dye is selected so as to produce significant emission whenexcited by the same excitation source or sources used to excite theother fluorescent dyes in the composition of interest.

For example, a polynucleotide sequencing reaction product mixture (chaintermination sequencing) may contain (1) four spectrally distinctfluorescent dyes, wherein each of the four dyes is correlated with adifferent polynucleotide base (e.g., fluorescently labeled dideoxysequencing) and (2) a monitoring dye that is spectrally distinct fromthe four other dyes. Movement of the monitoring dye in a separationchannel can be used to confirm that current flow and therefore properseparation of the sequencing reaction products is occurring. Monitoringdyes may be used in conjunction with sequencing reaction mixtures thatemploy either more or less than four dyes.

Another aspect of the invention is methods and compositions fordetecting the flow of electrical current through a separation channel ofa fluorescent polynucleotide separation apparatus. Such methods andcompositions are particularly useful with fluorescent polynucleotideseparation apparatus that employ multiple separation channels, e.g. amulti capillary or multiple microchannel system, because of thepossibility of failure of a subject separation channel. The subjectelectrical current flow monitoring methods involve the use offluorescent dyes that are spectrally distinct from fluorescently labeledpolynucleotides of primary interest. These spectrally distinctfluorescent dyes are referred to herein as monitoring dyes. In apreferred embodiment of the invention, the monitoring dye is selected soas to produce significant emission when excited by the same excitationsource or sources used to excite the other fluorescent dyes in thecomposition of interest.

For example, a polynucleotide sequencing reaction product mixture (chaintermination sequencing) may contain (1) four spectrally distinctfluorescent dyes, wherein each of the four dyes is correlated with adifferent polynucleotide base (e.g., fluorescently labeled dideoxysequencing) and (2) a monitoring dye that is spectrally distinct fromthe four other dyes. Movement of the monitoring dye in a separationchannel can be used to confirm that current flow and therefore properseparation of the sequencing reaction products is occurring. Monitoringdyes can be used in conjunction with sequencing reaction mixtures thatemploy either more or less than four dyes, e.g., one color or two colorbased sequencing.

Monitoring dyes may also be used in conjunction with other forms offluorescent polynucleotide fragment analysis in addition topolynucleotide sequencing. Such other forms of analysis include nucleicacid amplification products, ligation products, and the like.

The monitoring dyes may be used by themselves or may be conjugated toother molecules that can modify the migration rate of the monitoringdyes during electrophoresis, i.e., a mobility modifier. Examples of suchmigration modifying molecules include polynucleotides, polynucleotideanalogs, peptides, polypeptides, the mobility modifying moleculesdescribed in U.S. Pat. No. 5,514,543, and the like. Preferably, thesemobility modifying molecules are selected so as to not have spectralproperties that interfere with fluorescent detection of the dyes ofinterest. Detailed descriptions of how to conjugate fluorescent dyes tovarious compounds can be found in, among other places, Hermanson,Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996).Unless indicated otherwise by context of usage, the term “monitoringdye” includes monitoring dye conjugates.

Embodiments of the invention include compositions comprisingfluorescently labeled polynucleotides and one or more monitoring dyes,wherein the monitoring dyes are spectrally distinct from the otherfluorescent dyes in the mixture. The monitoring dyes may be added to thecomposition either before, after, or during the formation of thefluorescently labeled polynucleotides for analysis. For example, amonitoring dye may be added to a polynucleotide sequencing reactioneither before or after the reaction is terminated. In some embodimentsof the invention, the subject compositions comprise multiple differentmonitoring dyes. In such embodiments, the monitoring dyes are preferablyconjugates having different electrophoretic mobilities. In otherembodiments of the subject compositions, a single signal fluorescent dyeis present, but the dye molecules are conjugated to two or moredifferent mobility modifier species so as to produce multipleopportunities to detect the monitoring dye during electrophoreticseparation.

The invention also includes methods of detecting the flow of electricalcurrent through a separation channel of a fluorescent polynucleotideseparation apparatus by introducing a fluorescently labeledpolynucleotide composition into a channel of a fluorescentpolynucleotide separation apparatus. The fluorescently labeledpolynucleotide composition comprises a polynucleotide labeled with afirst fluorescent dye and a monitoring dye that is spectrally distinctfrom the first fluorescent dye. In most embodiments of the invention,the fluorescently labeled polynucleotide is a complex mixture ofdifferent length polynucleotides. Exemplary of such fluorescentlylabeled polynucleotide mixtures are the products of DNA sequencingreactions employing either fluorescently labeled primers orfluorescently labeled terminators, PCR amplification products formed byusing fluorescently labeled primers, fluorescently labeledmini-sequencing reactions, products, fluorescently labeledoligonucleotide ligation reaction products, and the like. Such reactionsproduce genetic information that may be analyzed in the fluorescentpolynucleotide separation apparatus. The monitoring dye is spectrallydistinct from the fluorescent dyes used to label the polynucleotidesthat convey genetic information. For example, the invention includes acomposition comprising a complex mixture of different fluorecentlylabeled polynucleotides produced from four color chain terminationsequencing and signal dye that is spectrally distinct from the fourfluorescent dyes on the different sequencing reaction products.

After the fluorescently labeled polynucleotide composition is introducedin the separation channel of a fluorescent polynucleotide separationapparatus, the apparatus is activated and the polynucleotide (and signaldyes, if not joined to a polynucleotide) permitted to separate along theseparation channel. The movement of the monitoring dye through theseparation channel may then be detected by the apparatus. Lack ofmovement of the monitoring dye (or dyes) or permutations of the movementof the monitoring dyes through the separation channels may be used todetect problems with the flow of electrical current through theseparation channel. The movement of monitoring dyes in differentchannels of a multiple channel fluorescent polynucleotide separationapparatus may be compared with one another so as to facilitate thedetection of problems with current flow.

Embodiments of the invention also include computer code for usingmonitoring dyes to monitor current flow in the subject methods, computerstorage media embodying such code, and programmable electronic computerprogrammed with such code.

The following example is intended to illustrate, and not limit, theinvention.

EXAMPLE 1

The data matrix, D, is essentially a table whose rows are theacquisition time points, and whose columns are the spectralbins/channels. This is schematically shown below in Table 1. TABLE 1 ARepresentation of the Data Matrix, D. Bin 1 Bin 2 Bin 3 . . . Bin k − 1Bin k T1 I11 I12 I13 . . . I1(k − 1) I1k T2 I21 I22 I23 . . . I2(k − 1)I2k T3 I31 I32 I33 . . . I3(k − 1) I3k . . . T(N − 1) 1(N − 1)1 I(N −1)2 I(N − 1)3 . . . I1(k − 1) I1k TN IN1 1N2 1N3 . . . I1(l − 1) I1k

The total emission profile is constructed by adding the intensities inall columns for each row. Table 2, below, shows a representation of thetotal emission profile. TABLE 2 Total Emission Profile of the DataMatrix, D. Total Emission T1 [I 11 + I 12 + I 13 + . + . + I 1(k − 1) +I 1k] T2 [I 21 + I 22 + I 23 + . + . + I 2(k − 1) + I 2k] T3 [I 31 + I32 + I 33 + . + . + I 3(k − 1) + I 3k] . . . T(N − 1) [I (N − 1) 1 + I(N − 1) 2 + I (N − 1) 3 + . + . + . I 1(k − 1) + I 1k] TN [I N1 + I N2 +I N3 + . + . + . I 1(k − 1) + I 1k]

The total emission profile represents peaks superimposed on background,as shown in FIG. 1 of the present application. The peaks in the totalemission profile are detected, and each peak's maximum referenced by itstime point, Tm, which corresponds to a particular row in Table 1. Thereference spectrum of each dye may be taken as the background-correctedsignal obtained in each spectral bin at the peak's maximum. For example,if the start of the peak (Ts) is taken as the background spectrum (peaksusually start in background), the corrected spectrum is the differencebetween row m and row s in Table 1. This is shown in Table 3, below.TABLE 3 Background-corrected Spectral Intensities (Peak maximum at pointm and background taken at point s) Bin 1 [I m1 − I s1] Bin 2 [I m2 − Is2] Bin 3 [I m3 − I s3] . . . Bin (k − 1) [I m (k − 1) − I s (k − 1)]Bin k [I m k − I s k]

EXAMPLE 2

A major advantage of fluorescent dye labeling is the ability tomultiplex short tandem repeat (STR) loci with different dyes andautomate the sequencing process. The ABI 377 is equipped to detect eachdye based on its emission spectrum. For example, four differentfluorescent dyes can be used to detect the bases in an mtDNA sequenceand the alleles of STR loci. These can include, for example, 5-FAM(blue), JOE (green), NED (yellow) and ROX (red). Each of the four dyesemits their maximum fluorescence at different wavelengths with someoverlap in the emission range.

A matrix file is a mathematical description of the spectral overlap,which is determined from the automated analysis of dye-labeled DNAfragments (matrix standard samples) for each of the four dyes. With thisinformation, the matrix file virtually instructs the sequencer to filterout the overlap, allowing the sequencer to distinguish between thesignals of each dye and display only one color for each base or alleleon an electropherogram.

In some cases, poor data collected can be successfully re-evaluatedusing newly created matrices.

To utilize a different dye set, one can perform a spectral calibrationusing an appropriate matrix standard (e.g., the DYEnamic ET matrixstandard for the ABI 3700 (Amersham Pharmacia Biotech)). This willcreate a new spectral calibration matrix for the new dye set.

EXAMPLE 3

A kit comprising dye standards can be used to calibrate the sequencedetection systems instruments. Particularly, the kit can be employed toestablish pure dye spectra and multi-component values on sequencinginstruments. Pure spectra information of the dye standards is collectedas part of the instrument installation and/or periodic maintenanceprocedure. The spectra data files are stored on a computer system andused by the sequencer application algorithm during data analysis.

EXAMPLE 4

FIG. 4 illustrates a data flow scheme, according to an embodiment of thepresent invention.

INCORPORATION BY REFERENCE

All publications, patent applications, and patents referenced in thespecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Equivalents

All publications, patent applications, and patents mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. Although only a fewembodiments have been described in detail above, those having ordinaryskill in the molecular biology art will clearly understand that manymodifications are possible in the preferred embodiment without departingfrom the teachings thereof. All such modifications are intended to beencompassed within the following claims. The foregoing writtenspecification is considered to be sufficient to enable skilled in theart to which this invention pertains to practice the invention. Indeed,various modifications of the above-described modes for carrying out theinvention which are apparent to those skilled in the field of molecularbiology or related fields are intended to be within the scope of thefollowing claims.

1. A method of monitoring a separation channel of a fluorescent polynucleotide separation apparatus, said method comprising: (i) introducing a fluorescently labeled polynucleotide composition to an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, said composition comprising (a) a polynucleotide labeled with a first fluorescent dye, and (b) a monitoring dye that is spectrally distinct from the first fluorescent dye; (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel; and (iii) detecting for the monitoring dye at one or more regions downstream of said inlet end; whereby detection of the monitoring dye at said one or more regions is indicative of flow along the channel.
 2. The method of claim 1, wherein the fluorescently labeled polynucleotide composition comprises: a plurality of polynucleotides comprising at least two polynucleotides labeled with at least two respective spectrally distinct fluorescent dyes, wherein the monitoring dye is spectrally distinct from each of the at least two spectrally distinct fluorescent dyes.
 3. The method of claim 2, wherein the plurality of polynucleotides labeled with at least two spectrally distinct fluorescent dyes comprises a polynucleotide sequencing reaction product mixture.
 4. The method of claim 1, wherein the monitoring dye is attached to a polynucleotide.
 5. The method of claim 1, wherein the monitoring dye produces significant emission when excited by the same excitation source or sources used to excite other fluorescent dyes of the fluorescently labeled polynucleotide composition.
 6. The method of claim 1, further comprising detecting current in the separation channel by detecting migration of the monitoring dye.
 7. The method of claim 1, further comprising detecting separation of the sequencing reaction products by detecting migration of the monitoring dye.
 8. A method of monitoring a separation channel of a fluorescent polynucleotide separation apparatus, said method comprising: (i) introducing a fluorescently labeled polynucleotide composition to an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, said composition comprising (a) at least two spectrally distinct fluorescent dyes, wherein each of the at least two dyes is correlated with a different polynucleotide base, and (b) a monitoring dye that is spectrally distinct from each of the at least two spectrally distinct fluorescent dyes; (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel; and (iii) detecting for the monitoring dye at one or more regions downstream of said inlet end; whereby detection of the monitoring dye at said one or more regions is indicative of flow along the channel.
 9. The method of claim 8, wherein the fluorescently labeled polynucleotide composition comprises a polynucleotide sequencing reaction product mixture.
 10. The method of claim 8, wherein the monitoring dye is attached to a polynucleotide.
 11. The method of claim 8, wherein the monitoring dye produces significant emission when excited by the same excitation source or sources used to excite the at least two spectrally distinct fluorescent dyes of the fluorescently labeled polynucleotide composition.
 12. The method of claim 8, further comprising detecting current in the separation channel by detecting migration of the monitoring dye.
 13. The method of claim 8, further comprising detecting separation of the sequencing reaction products by detecting migration of the monitoring dye.
 14. The method of claim 8, wherein the at least two spectrally distinct fluorescent dyes comprise four spectrally distinct fluorescent dyes.
 15. The method of claim 8, wherein the at least two spectrally distinct fluorescent dyes comprise at least four spectrally distinct fluorescent dyes.
 16. A computer readable medium having computer code for detecting a monitoring dye at one or more regions downstream of an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, during a method comprising: (i) introducing a fluorescently labeled polynucleotide composition to the inlet end, the composition comprising (a) at least two spectrally distinct fluorescent dyes, wherein each of the at least two dyes is correlated with a different polynucleotide base, and (b) the monitoring dye, the monitoring dye being spectrally distinct from each of the at least two spectrally distinct fluorescent dyes; and (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel, wherein detection of the monitoring dye at the one or more regions is indicative of flow along the channel.
 17. The computer readable medium of claim 16, further comprising computer code for detecting current in the separation channel by detecting migration of the monitoring dye.
 18. The computer readable medium of claim 16, further comprising computer code for detecting separation of sequencing reaction products by detecting migration of the monitoring dye.
 19. The computer readable medium of claim 16, further comprising: computer code for causing a computer to compare a signal-to-noise ratio of a first dye to a minimum signal-to-noise ratio; and computer code for causing a computer to determine whether the signal-to-noise ratio of the first dye exceeds the minimum signal-to-noise ratio.
 20. An electronic computer comprising the computer readable medium of claim 16 and programmed with the computer code. 